This is an application for renewal of funding of work that is exploring chemical sensing using Paramecium as a chemosensory cell. This organism presents the opportunity to apply multiple techniques in addressing hypotheses: biochemistry, molecular biology, transmission genetics, electrophysiology behavioral analysis. While cellular and molecular genetic approaches to some extent must disrupt the intact receptor cell or impose unphysiological conditions, it is often possible with Paramecium to return the study to the whole cell and ask what physiologically relevant changes have been made in chemosensory signal transduction. In the past funding period, we have adopted antisense technology to our study of the interaction between transduction components. Receptors in at least one pathway in Paramecium appear to activate the plasma membrane calcium pump, creating the sustained hyperpolarizing conductance that characterizes attractants. We have made use of antisense oligodeoxynucleotides (ODNs) for calmodulin, a regulator of the pump, to down regulate the levels of calmodulin in order to indirectly inhibit the pump as a test of its role in chemoresponse. We provided evidence that a conductance known to be calmodulin regulated is reduced in the antisense treated cells. More importantly, we have shown that chemosensory behavior is correspondingly and selectively inhibited. Response to acetate that initiates a pathway, thought to involve the pump, is inhibited while response to NH4Cl, utilizing a pathway without the pump, is not inhibited. We have prepared the plasmids necessary to stably transform cells to improve our physiological studies of these cells and we have developed the antibodies and immunocytochemistry that are necessary to monitor the transformation of cells with antisense ODNs or plasmids with genes for which there is no obvious behavioral, selectable phenotype. Other tests of interactions of transduction components using immunoprecipitation are on-going. We propose to test three related hypotheses: 1) Receptors for at least one of the three signal transduction pathways are coupled to the calcium pump. 2) Receptors, the calcium pump and other transduction components interact and are found within the same membrane domain. 3) The calcium pump and cyclases couple to GPI anchored receptors and possibly G proteins. We will use antisense and other transformation methods to produce phenocopies of mutants in the genes for the transduction components; confocal microscopy to locate components of the transduction pathways; perturbation of GPI anchored proteins by phospholipase C treatment.